Immunocytochemistry of soluble and non-soluble nuclear proteins on squash preparations of mammalian meiotic cells.
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Biomedical subjects
R Benavente
Publications and source records attributed to R Benavente.
Stage-specific gene expression during rat spermatogenesis: application of the mRNA differential display method.
Spermatogenesis is a complex differentiation process which requires the coordinate synthesis of diverse stage-specific proteins. In attempting a large-scale identification and characterization of those proteins, we have made use of the recently described mRNA differential display method (Liang and Pardee, Science 257: 967-971, 1992). This method is based on the reverse transcription of mRNAs obtained from two different cell populations (pachytene spermatocytes and spermatids in the present study) followed by a PCR reaction and comparison of the individual cDNA populations in a polyacrylamide gel system. Up to the present we have been able to identify 268 cDNA bands. Most of them (77%) are common to both cell stages. From the differentially expressed bands (23%) an ample majority was spermatid-specific (74%). According to our present results we conclude that the mRNA differential display is a promising approach for investigations on stage-specific gene expression during a differentiation process like spermatogenesis.
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Cytochemical and immunocytochemical characterization of kinetochores in the holocentric chromosomes of Graphosoma italicum.
In the present paper we have analyzed the trilaminar kinetochores present in the mitotic holocentric chromosomes of the heteropteran Graphosoma italicum by means of different cytochemical and immunocytochemical techniques at the ultrastructural level. The results obtained after EDTA and osmium tetroxide/p-phenylenediamine, both cytochemical methods for preferential detection of ribonucleoproteins, show that the inner and outer kinetochore layers are selectively contrasted. These results agree with those previously reported for the kinetochores of monocentric chromosomes. Additionally, using cytochemical techniques such as ethanolic phosphotungstic acid staining and silver impregnation, which recognize proteins, we report a different response of the inner kinetochore layer compared with those obtained in the middle and outer layers. Finally, our immunoelectron microscopic results employing monoclonal antibodies against DNA indicate the absence of this component from the kinetochore plates in holocentric chromosomes. Although the trilaminar appearance of kinetochores in holocentric chromosomes is very similar to that of the localized kinetochores in mammals, our observations imply the existence of significant differences on the macromolecular organization of both types of kinetochores.
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An Mr 51,000 protein of mammalian spermatogenic cells that is common to the whole XY body and centromeric heterochromatin of autosomes.
During mammalian male meiotic prophase the sex chromosomes form a structure called the XY body or sex vesicle. This structure is characterized by differential condensation of chromatin and transcriptional inactivity. The reasons and mechanisms for the allocyclic behaviour of sex chromosomes with respect to autosomes are largely unknown. In order to gain insight into the process of XY-body formation we are involved in the characterization of proteins associated with meiotic sex chromosomes by immunological approaches. Here we report on the identification of an Mr 51,000 protein (p51) that is homogeneously distributed in the XY body of rodents as shown by immunocytochemistry with the novel monoclonal antibody 4EC. Interestingly, in germ line cells the antibody also labelled the centromeric heterochromatin of autosomes. We speculate that p51 may be a component of the mechanisms that lead to wide chromosome regions becoming inaccessible for transcription and/or recombination events.
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Immunocytochemical localization of DNA in synaptonemal complexes of rat and mouse spermatocytes, and of chick oocytes.
The distribution of DNA in synaptonemal complexes of rat and mouse spermatocytes, and of chick oocytes was investigated by immunogold electron microscopy. Except for a few specific sites, DNA was not immunolocalized in the space between lateral elements of the complex. Some labeled fibrils connecting the lateral elements with the central element were observed associated with recombination nodules or near them. However, other labeled fibrils in the space between lateral elements did not appear to present any relationship to recombination nodules. The immunocytochemical approaches used here confirmed the presence of significant amounts of DNA in the lateral elements as previously indicated by preferential DNA staining methods. Furthermore, our findings support the view that recombination nodules are the site of chiasma formation.
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Presence of a nuclear lamina in pachytene spermatocytes of the rat.
The nuclear lamina is a karyoskeletal structure located at the periphery of cell nuclei. The major constituents are the lamins, which belong to the evolutionarily conserved multigene family of intermediate filament proteins. Lamins show a conspicuous cell type-specific expression pattern. The majority of somatic cells of vertebrates express A-type (lamins A and C) as well as B-type (lamins B1 and B2) lamins. Although a lamina structure has been demonstrated to be a ubiquitous component of somatic nuclei its existence in certain meiotic stages during spermatogenesis has been a matter of debate. In this study, we investigated the expression of lamins in rat spermatogenic cells using immunological and protein-chemical methods. We report on the presence of a nuclear lamina structure in rat pachytene spermatocytes. With the aid of a novel broad-reacting lamin antibody we have demonstrated the expression of a protein that is closely related, if not identical, to lamin B1.
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The DNA topoisomerase I inhibitor camptothecin blocks postmitotic reformation of nucleoli in mammalian cells.
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Meiosis-specific protein selectively associated with sex chromosomes of rat pachytene spermatocytes.
During the first meiotic prophase of mammalian spermatogenesis, the sex chromosomes X and Y show a characteristic allocyclic behavior with respect to the autosomes. This is particularly evident during pachytene stage when sex chromosomes form the so-called sex vesicle. This structure is characterized by the condensed state of chromatin, transcriptional inactivity, and the limited extension of chromosome pairing, which is usually restricted to a short segment of sex chromosome axial elements. The molecular basis and functional significance of sex vesicle formation during mammalian spermatogenesis remain obscure. Here we report on the identification of a meiosis-specific sex vesicle protein we called XY40. Immunocytochemical localization on rat testis cryosections with a XY40-specific monoclonal antibody revealed that the labeling is confined to the axial elements of sex chromosomes. Biochemical characterization showed that protein XY40 (40 kDa; pI 5.7-5.8) can be extracted from rat pachytene spermatocytes and recovered in particles of 9.5 S with a native molecular mass of approximately 152 kDa. We speculate that protein XY40 may be involved in the allocyclic behavior of sex chromosomes during male meiotic prophase.
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Identification of a structural protein component of rat synaptonemal complexes.
Synaptonemal complexes (SCs) are evolutionarily conserved nuclear structures of meiotic cells which form during the zygotene stage of the first meiotic prophase and are responsible for the pairing of homologous chromosomes. Their formation appears to be a prerequisite for crossing-over events and proper chromosome segregation during the first meiotic division. Despite knowledge of their central role in genetic recombination processes very little is known about the molecular composition and the mechanisms governing the assembly of the SCs. In the present study we report on the characterization of a monoclonal antibody (SC14f10) which enabled us to identify a novel SC protein termed SC48. Protein SC48 has a Mr of 48,000 and migrates in two-dimensional gels with a pH value of 6.9. By means of immunogold EM we localized this protein to the central region of the SC. In cell fractionation experiments we recovered protein SC48 together with SC-residual structures in a karyoskeletal fraction of pachytene spermatocytes. Our results indicate that SC48 is a meiosis-specific structural protein component of the SC probably involved in the pairing of homologous chromosomes.
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Identification of a short nuclear lamin protein selectively expressed during meiotic stages of rat spermatogenesis.
The nuclear lamina is a karyoskeletal structure located at the nuclear periphery and intimately associated with the inner nuclear membrane. It is composed of a multigene family of proteins, the lamins, which show a conspicuous cell type-specific expression pattern. The functional role of lamins has not been definitively established but available information indicates that they are involved in the organization of nuclear envelope and interphase chromatin. Spermatogenesis is characterized, among other features, by stage-specific changes in chromatin organization and function. These changes are accompanied by modifications in the organization and composition of the nuclear lamina. In previous experiments we have determined that rat spermatogenic cells express a lamin closely related, if not identical, to lamin B1 of somatic cells; whereas rat somatic lamins A, C, D and E were not detected. Considering that chromatin reorganizations during spermatogenesis may be directly or indirectly related to changes of the nuclear lamina we have decided to further investigate lamin expression during this process. Here we report on the identification of a 52 kDa protein of the rat which, according to immunocytochemical and biochemical data, appears to be a novel nuclear lamin. Using meiotic stage-specific markers, we have also demonstrated that this short lamin is selectively expressed during meiotic stages of spermatogenesis.
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Postmitotic nuclear reorganization events analyzed in living cells.
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Functional and dynamic aspects of the mammalian nucleolus.
Nucleoli are the sites of ribosome biogenesis. Transcription of the ribosomal RNA genes as well as processing and initial packaging of their transcripts with ribosomal and non-ribosomal proteins all occur within the nucleolus in an ordered manner and under defined topological conditions. Components of the nucleolus have been localized by immunocytochemistry and their functional aspects investigated by microinjection of antibodies directed against the enzyme responsible for rDNA transcription, RNA polymerase I. The role of nascent transcripts in postmitotic formation of nucleoli will be discussed.
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Functional role of newly formed pore complexes in postmitotic nuclear reorganization.
Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK2 cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK2 cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophase-like completed cytokinesis, their nuclei showed a telophase-like organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.
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Nucleocytoplasmic sorting of macromolecules following mitosis: fate of nuclear constituents after inhibition of pore complex function.
PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68,000 pore complex glycoprotein) or of wheat germ agglutinin (WGA) complete cytokinesis. However, their nuclei remain stably arrested in a telophase-like organization characterized by highly condensed chromatin and the absence of nucleoli, indicating a requirement for pore-mediated transport for the reassembly of interphase nuclei. We have now examined this requirement more closely by monitoring the behavior of individual nuclear macromolecules in microinjected cells using immunofluorescence microscopy and have investigated the effect of microinjecting the antibody or WGA on cellular ultrastructure. The absence of nuclear transport did not affect the sequestration into daughter nuclei of components such as DNA, DNA topoisomerase I and the nucleolar protein fibrillarin that are carried through mitosis on chromosomes. On the other hand, lamins, snRNAs and the p68 pore complex glycoprotein, all cytoplasmic during mitosis, remained largely cytoplasmic in the telophase-arrested cells. Electron microscopy showed the nuclei to be surrounded by a double-layered membrane with some inserted pore complexes. In addition, however, a variety of membranous structures with associated pore complexes was regularly noted in the cytoplasm, suggesting that chromatin may not be essential for the postmitotic formation of pore complexes. We propose that cellular compartmentalization at telophase is a two-step process. First, a nuclear envelope tightly encloses the condensed chromosomes, excluding non-selectively all macromolecules not associated with the chromosomes. Interphase nuclear organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm.
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Monoclonal antibodies to a Mr 68,000 pore complex glycoprotein interfere with nuclear protein uptake in Xenopus oocytes.
Using a monoclonal antibody (PI1) raised against mouse lymphocyte nuclear matrix fractions we have identified a N-acetylglucosamine (GlcNAc)-containing glycoprotein of Mr 68,000 as a component of the nuclear pore complexes of Xenopus laevis oocytes. The antigenic determinant recognized by antibody PI1 comprises both the sugar moiety and protein sequences since, on the one hand, added GlcNAc competed effectively for antibody binding and, on the other hand, the antibody reacted in immunoblots with only one member of the GlcNAc-containing pore complex glycoprotein family. By using immunogold-electron microscopy we could demonstrate that the Mr 68,000 glycoprotein was located preferentially to the cytoplasmic side of the pore complex channel. When radiolabeled soluble nuclear proteins were injected into the cytoplasm of Xenopus oocytes, their reentry into the nucleus was almost completely inhibited in the presence of antibody PI1 as shown by two-dimensional gel electrophoresis. The results indicate that the evolutionarily conserved Mr 68,000 glycoprotein is involved in transport processes of karyophilic proteins from the cytoplasm into the nucleus.
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Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells.
After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.
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Identification and definition of nucleolus-related fibrillar bodies in micronucleated cells.
Small nucleolus-related bodies which occur in the nucleoplasm of "micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.
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Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.
The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.
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